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Contribution of genetic and environmental circadian disruptions to the development of comorbid behavioral and metabolic deficits in mice
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Untersuchungen zur Pathophysiologie der PRDM12-assoziierten angeborenen Schmerzunempfindlichkeit
Get PDFDas Schmerzempfinden ist unerlässlich für die Aufrechterhaltung der Integrität des Körpers. Während die molekularen Mechanismen, die die Differenzierung der verschiedenen Subtypen nozizeptiver Neurone steuern, zunehmend verstanden werden, sind die Faktoren, die bei der Initiierung der nozizeptiven Linie entscheidend sind, noch weitgehend unbekannt. Biallelische Varianten im humanen PRDM12-Gen verursachen eine seltene autosomal-rezessiv erbliche Erkrankung, bei der es zu einem angeborenen Fehlen des Schmerzempfindens kommt (congenital insensitivity to pain, CIP). Andere sensible Modalitäten (leichte Berührung, Vibration und Propriozeption), autonome Funktionen sowie Geruchs- und Hörsinn sind nicht eingeschränkt. Nervenbiopsien betroffener Patienten zeigen einen deutlichen Verlust von Aδ-Fasern, Hautbiopsien ein vollständiges Fehlen freier dermaler Nervenendigungen.
Um zu klären, ob diese Veränderungen auf einen Defekt in der Entwicklung oder eine rasche Degeneration sensibler Neurone zurückzuführen sind, wurde in Kooperation mit der Arbeitsgruppe von Herrn Professor Roman Chrast (Karolinska Institutet, Stockholm, Schweden) ein Mausmodell etabliert, bei dem die Expression des Prdm12-Gens ausgeschaltet wurde. Spinalganglien PRDM12-defizienter Mausembryonen waren im Vergleich zu den Spinalganglien von Wildtyp-Embryonen deutlich kleiner und wiesen weniger Neurone auf. Die weitergehende Untersuchung des Mausmodells durch die Arbeitsgruppe von Herrn Professor Roman Chrast zeigte, dass die beobachtete Größenminderung und verminderte Zellzahl der Spinalganglien auf ein Ausbleiben der Entwicklung der nozizeptiven Linie zurückzuführen ist. Die Entwicklung von Neuronen für andere sensible Modalitäten war hingegen nicht beeinträchtigt.
Einige Mitglieder der PRDM-Proteinfamilie, zu der auch PRDM12 gehört, sind Transkriptionsrepressoren und rekrutieren Co-Transkriptionsfaktoren, um chromatinregulierende Multiproteinkomplexe zu bilden. Unter Verwendung eines heterologen Promotors konnte gezeigt werden, dass PRDM12 ebenfalls dosisabhängig als Transkriptionsrepressor wirkt, wobei diese Funktion durch krankheitsassoziierte PRDM12-Varianten teilweise aufgehoben wird. Mit Hilfe von Immunpräzipitationsstudien konnte der Co-Transkriptionsfaktor CBFA2T2 als neuer potentieller Interaktionspartner von PRDM12 identifiziert werden. Passend zur angenommenen Interaktion überlappen die zeitlichen und räumlichen Expressionsmuster von Prdm12 und Cbfa2t2, und beide Proteine kolokalisieren in distinkten Strukturen im Zellkern. Darüber hinaus zeigen PRDM12 und CBFA2T2 einen synergistischen Effekt im Sinne einer verstärkten Repression eines heterologen Promotors.
Die Ergebnisse dieser Arbeit weisen auf eine spezifische und essenzielle Funktion von PRDM12 für die Initiierung der gesamten nozizeptiven Linie hin. Mechanistisch scheint PRDM12 als Transkriptionsrepressor zu wirken, möglicherweise in einem Komplex mit dem hier identifizierten Bindungspartner CBFA2T2. In Anbetracht der entscheidenden Rolle von PRDM12 in der Entwicklung von Nozizeptoren ist davon auszugehen, dass die weitergehende Aufklärung der beteiligten molekularen Mechanismen wesentlich zu einem besseren Verständnis der Regulation der sensiblen Neurogenese beitragen wird.The sensation of pain is essential for maintaining the integrity of the body. While the molecular mechanisms that control the differentiation of the various subtypes of nociceptive neurons are increasingly understood, the factors that are critical in initiating the nociceptive lineage are still largely unknown. Biallelic variants in the human PRDM12 gene cause a rare autosomal recessive inherited disorder in which there is a congenital absence of pain sensation (congenital insensitivity to pain, CIP). Other sensory modalities (light touch, vibration and proprioception), autonomic functions, sense of smell and hearing are not impaired. Nerve and skin biopsies of affected patients show a marked loss of Aδ-fibres, skin biopsies a complete absence of free dermal nerve endings.
To clarify whether these changes are due to a defect in the development or a rapid degeneration of sensory neurons, a mouse model was established in cooperation with the research group of Professor Roman Chrast (Karolinska Institutet, Stockholm, Sweden) in which the expression of the Prdm12 gene was switched off. Spinal ganglia of PRDM12-deficient mouse embryos were significantly smaller and had fewer neurons compared to the spinal ganglia of wild-type embryos. Further investigation of the mouse model by Professor Roman Chrast's research group showed that the observed reduction in size and decreased cell number of the spinal ganglia was due to a failure of the nociceptive lineage to develop. In contrast, the development of neurons for other sensory modalities was not affected.
Some members of the PRDM protein family, which includes PRDM12, are transcriptional repressors and recruit co-transcription factors to form chromatin-regulating multiprotein complexes. Using a heterologous promoter, PRDM12 was also shown to act as a transcriptional repressor in a dose-dependent manner, while this function is partially abrogated by disease-associated PRDM12 variants. With the help of immunoprecipitation studies, the co-transcription factor CBFA2T2 could be identified as a new potential interaction partner of PRDM12. Consistent with the hypothesised interaction, the temporal and spatial expression patterns of Prdm12 and Cbfa2t2 overlap, and both proteins colocalise in distinct structures in the nucleus. Furthermore, PRDM12 and CBFA2T2 show a synergistic effect in terms of enhanced repression of a heterologous promoter.
The results of this work indicate a specific and essential function of PRDM12 for the initiation of the entire nociceptive lineage. Mechanistically, PRDM12 appears to act as a transcriptional repressor, possibly in complex with the binding partner CBFA2T2 identified here. Considering the crucial role of PRDM12 in the development of nociceptors, it can be expected that further elucidation of the molecular mechanisms involved will contribute significantly to a better understanding of the regulation of sensory neurogenesis
Generation of oligodendrocytes and characterisation of their role in axonal support
Get PDFGlial cells, comprising of oligodendrocytes, astrocytes and microglia, have come a long way from their simplistic description as nerve glue. Today, we know that neuron-glia interactions are a fundamental aspect of neuronal function and brain homeostasis. Oligodendrocytes (OLGs) are the myelinating glia of the central nervous system forming myelin sheaths that enwrap axons. These cells are involved in axonal maintenance and survival, neuronal circuitry adaptation and immunomodulation. However, many aspects of oligodendrocyte physiology and pathology are still not completely understood. Factors that have contributed to the knowledge gaps associated with these cells include the complicated and inefficient protocols for the isolation of primary cells, the lack of defined stage specific markers and of adequate transgenic tools that would allow the manipulation of these cells in vitro.
In this thesis I conducted a comparative study to evaluate the differentiation efficiency of OLGs derived from human induced pluripotent stem cells (iPSCs) via the ectopic expression of oligodendrocyte transcription factors. I aimed to provide benchmarking criteria for the reproducibility and robustness of iPSC derived OLGs (iOLGs) protocols. I observed that iPSCs overexpressing solely SOX10 differentiated, at low yields, into O4 and MBP expressing iOLGs, while overexpression of a combination of three transcription factors, SOX10, OLIG2 and NKX6.2 (SON) lead to a higher differentiation efficiency. By including a purification step I significantly improved oligodendrocyte differentiation yields and reduced the population heterogeneity. The gene expression profile of SON-induced iOLGs confirmed the expression of oligodendrocyte differentiation markers. Furthermore, using a co-culture platform with iPSC-derived neurons, astrocytes and microglia we showed that these cells can migrate within the culture and form myelin-like structures in vitro.
In this thesis I also aimed to address the wider knowledge gap of the mechanism of axonal support by myelinating oligodendrocytes. Oligodendrocytes secrete ferritin heavy chain (FTH1) protein, which may be internalised by neighbouring neurons and act as an antioxidant defence system by storing and detoxifying the excess of neuronal intracellular iron. Interestingly, Fth1 mRNA is among the three most highly abundant transcripts found in purified myelin, despite not behaving like a myelin-resident protein. Given the importance of FTH1 protein for neuronal protection it is reasonable to expect that the targeted transport and local translation of Fth1 mRNA could effectively provide a way for oligodendrocytes to rapidly respond to external stimuli. As such I aimed to characterise the nature of the Fth1 transcript in oligodendrocytes. Fth1 mRNA shows the characteristic granular distribution along the distal processes, reminiscent of other locally repressed mRNAs in both mouse and human oligodendrocytes. Moreover, Fth1 mRNA is not associated with processing bodies or stress granules in mature oligodendrocytes, instead it appears to be a unique type of cytoplasmic RNA. To identify the RNA-binding proteins that promote Fth1 mRNA localization or translation repression, I developed a proteomics approach to selectively isolate native RNA-protein complexes. I identified 19 potential protein candidates that could be associated with Fth1 mRNA translocation and/or translation repression in oligodendrocytes.
In conclusion, our study is the first step to establish a standardised method for oligodendrocyte differentiation via ectopic transcription factor expression. Indeed, continuous improvement of the established protocols will allow the development of reproducible and cost-effective human iPSC-derived oligodendrocyte models and ultimately facilitate the utilisation of the iPSC technology to study the mechanisms and pathways of human OLG migration and myelination. In addition, our study also suggests the existence of a previously unknown RNA trafficking mechanism for Fth1 mRNA and local protein translation in myelin. We also identified potential Fth1 mRNA binding proteins that could provide additional insights into the impact of neuronal cues in the regulation of FTH1 protein expression and secretion by oligodendrocytes
Technische Umsetzung der digitalen Infrarotfotografie zur Diagnostik von Erkrankungen des Pferdeauges
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Epigenetic reprogramming of pancreatic cancer cells as a new therapeutic option
Get PDFLarge-scale gene expression analyses have demonstrated that pancreatic ductal adenocarcinoma can be classified into different molecular subtypes with clinical significance (Collisson et al., 2011, Moffitt et al., 2016, Bailey et al., 2016). So far, great effort has been put into unveiling the factors responsible for tumor heterogeneity in pancreatic cancer. Since epigenetic modifiers are, besides the four driver gene mutations KRAS, p16, p53, and SMAD4, among the most frequently mutated genes in PDAC, this study aimed at investigating the role of epigenetic changes in the two molecular PDAC cancer subtypes, represented by a classical and basal phenotype, as well as their therapeutic potential in pancreatic cancer cell lines (Bailey et al., 2016).
The data showed that subtype-specific gene expression of cellular differentiation marker genes, such as EpCAM and GATA6, is epigenetically regulated. Chromatin-immunoprecipitation results demonstrated that the expression of these epithelial differentiation marker genes is activated through histone acetylation marks in the classical subtype. In contrast, their expression is repressed in the basal or quasimesenchymal subtype through increased levels of histone ubiquitination as well as a loss of histone acetylation marks. DNA methylation seemed to only play a minor part in regulating subtype-specific gene expression profiles of EpCAM and GATA6.
Despite subtype-specific histone acetylation levels, single-drug treatment with chemical inhibitors targeting histone acetylation and deacetylation marks only showed limited effects in vitro. Classical and basal PDAC cell lines were almost completely resistant to HAT inhibitor treatment with A485. Only one of the basal cell lines, MIAPaca-2, reached a 50 % survival rate at the maximum dosage of 10 µM A485 (see Figure 12A, left panel). High doses of the HDAC inhibitor Vorinostat were able to inhibit cell survival to a greater extent, but the response was independent of the transcriptomic subtypes. It is possible that a compensatory upregulation of other epigenetic modifications limits the therapeutic effects. Hence, a multiplex CRISPR/Cas9 knockout plasmid targeting a combination of epigenetic modifiers (HDAC2, DNMT3A, RING1B) was constructed to induce simultaneous genetic knockout of all three target genes. However, transfection of a basal pancreatic cancer cell line with this plasmid did not yield a successful knockout cell line. Most likely, the combinatory knockout impaired critical cellular functions to such an extent that cell death occurred. To overcome the limitations of a multiplex CRISPR/Cas9 knockout strategy, a successive knockout of one target gene after the other might be a more successful strategy to analyze the effect of a combinatory loss of different epigenetic modifiers. Furthermore, a selection marker should be included in the plasmids to ensure successful transfection. The generated knockout cell line can then be used for transcriptome analysis by RNA sequencing as well as for basic cell assays and drug sensitivity tests.
In order to translate preclinical data with epigenetic inhibitors into successful clinical trials, further studies are needed to determine subtype-specific changes after epigenetic targeting. For instance, unpublished data within the working group showed that HAT inhibitor treatment of cell lines with a classical PDAC subtype strongly downregulated the expression of GATA6 and decreased Gemcitabine drug sensitivity indicating a poorer outcome. These results emphasize the importance of establishing patient stratification systems in order to maximize the success of current treatment strategies.
Overall, this thesis showed that the transcriptomic profiles defining molecular PDAC subtypes are in part regulated through epigenetic modifications. Although the targeting of single epigenetic modifiers showed some success in tumor cell reprogramming, the therapeutic targeting with epigenetic drugs remains limited. Thus, the precise effects of combination therapies with multiple epigenetic inhibitors need further investigation